Preparation of UP-nanovaccine from Cell Lysate

Vx3 protein was obtained and covalently linked to α-Al₂O₃ nanoparticles to generate α-Al₂O₃-Vx3 nanoparticles according to our previous reports (12 (link), 13 (link)). 4T1/WT cells and 4T1/EPB cells were treated with 200 nM bortezomib (Millennium Pharmaceuticals, USA) and 20 mM NH₄Cl (Sigma, USA) for 9 h. The cells were collected and lysed in RIPA lysis buffer (Millipore, USA) containing protease inhibitors (MedChemExpress, USA), phosphatase inhibitors (MedChemExpress, USA), and PR-619 (MedChemExpress, USA). The cell lysate was reacted with α-Al₂O₃-Vx3 nanoparticles under stirring for 12 h at 4°C to generate α-Al₂O₃-UPs nanovaccine (named UP-nanovaccine) according to our previous study (13 (link), 20 (link)). The precipitates (UP-nanovaccine) were collected by centrifugation (12,000 g, 30 min, 4°C), and the supernatant was collected as unbound lysate, followed by the detection of ubiquitin protein levels in the three samples using Western blotting. The covalently linked product UP-nanovaccine was collected by centrifugation, and the number of UPs enriched by α-Al₂O₃-Vx3 was evaluated by collecting and calculating the difference between the number of UPs in the supernatant before and after the reaction by BCA Protein Assay Kit (Beyotime Biotechnology, China) according to the protocol of the manufacturer.